We describe a novel synthetic N-glycosylation pathway to produce recombinant proteins carrying human-like N-glycans in Saccharomyces cerevisiae, at the same time addressing glycoform and glycosylation efficiency. The Δalg3 Δalg11 double mutant strain, in which the N-glycans are not matured to their native high-mannose structure, was used. In this mutant strain, lipid-linked Man(3)GlcNAc(2) is built up on the cytoplasmic side of the endoplasmic reticulum, flipped by an artificial flippase into the ER lumen, and then transferred with high efficiency to the nascent polypeptide by a protozoan oligosaccharyltransferase. Protein-bound Man(3)GlcNAc(2) serves directly as a substrate for Golgi apparatus-targeted human N-acetylglucosaminyltransferases I and II. Our results confirmed the presence of the complex human-like N-glycan structure GlcNAc(2)Man(3)GlcNAc(2) on the secreted monoclonal antibody HyHEL-10. However, due to the interference of Golgi apparatus-localized mannosyltransferases, heterogeneity of N-linked glycans was observed.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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Evidence ID | Analyze ID | File | Description |
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